玉米花色苷含量相关状和铁Fe、锌Zn含量相关状的QTL初步定位及玉米籽粒花色苷含量两个主效QTLAC6和AC0的精细定位和上位研究.doc

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1、【精品文档】如有侵权，请联系网站删除，仅供学习与交流玉米花色苷含量相关状和铁Fe、锌Zn含量相关状的QTL初步定位及玉米籽粒花色苷含量两个主效QTLAC6和AC0的精细定位和上位研究.精品文档.单位代码 学 号 博士学位论文玉米花色苷含量相关性状和铁 (Fe)、锌 (Zn)含量相关性状的QTL初步定位及玉米籽粒花色苷含量两个主效QTLAC6和AC10的精细定位和上位性研究论文作者：覃鸿妮指导教师：蔡一林学科专业：作物遗传育种研究方向：玉米分子育种提交论文日期：论文答辩日期：学位授予单位：中 国 重 庆2012年3月Dissertation for Doctors Degree of South

2、west UniversityQTL analysis of anthocyanin-related traits and Fe and Zn concentration-associated traitsand fine mapping and analyzing epistatic gene effects of two genes AC6 and AC10 that controls anthocyanin content in maize(zea mays L)kernelDoctors degree candidate: Supervisor: Prof. :Major: Direc

3、tion: ChongqingChinaMarch, 2012独创性声明学位论文题目： 本人提交的学位论文是在导师指导下进行的研究工作及取得的研究成果。论文中引用他人已经发表或出版过的研究成果，文中已加了特别标注。对本研究及学位论文撰写曾做出贡献的老师、朋友、同仁在文中作了明确说明并表示衷心感谢。学位论文作者： 签字日期： 年 月 日学位论文版权使用授权书本学位论文作者完全了解西南大学有关保留、使用学位论文的规定，有权保留并向国家有关部门或机构送交论文的复印件和磁盘，允许论文被查阅和借阅。本人授权西南大学研究生院（筹）可以将学位论文的全部或部分内容编入有关数据库进行检索，可以采用影印、缩印或扫

4、描等复制手段保存、汇编学位论文。（保密的学位论文在解密后适用本授权书，本论文：不保密，保密期限至 年 月止） 。学位论文作者签名： 导师签名：签字日期： 年 月 日 签字日期： 年 月 日目 录摘 要IABSTRACTV第一章 文献综述11.1数量性状基因座（QTL）定位11.1.1QTL 主要作图方法11.1.2QTL 定位方法最新进展21.2玉米基因组学研究41.2.1结构基因组学41.2.2比较基因组学51.2.3功能基因组学61.2.4生物信息学61.3花色苷研究进展71.3.1花色苷结构和代谢途径71.3.2花色苷生理功能81.3.3控制花色苷合成的结构基因和调节基因91.4植物体内

5、矿质元素的代谢平衡101.4.1植物对矿质元素耐受能力的研究进展101.4.2作物铁 (Fe) 和锌 (Zn) 生物强化研究进展12第二章 引言132.1研究目的与意义132.2技术路线14第三章 玉米中色素及矿质元素相关性状的QTL定位153.1材料与方法153.1.1试验材料153.1.2试验方法163.2结果与分析213.2.1表型分析213.2.2遗传连锁图谱构建323.2.3QTL定位383.3讨论543.3.1不同环境（重庆和云南）检测到的QTL比较543.3.2不同群体（MuS和MoS）检测到的QTL比较553.3.3QTL的成簇分布及与前人QTL定位研究结果的比较57第四章 玉

6、米籽粒中花色苷含量主效QTL-AP6的精细定位及候选基因预测594.1材料与方法594.1.1试验材料594.1.2试验方法594.2结果与分析614.2.1AC6近等基因系的构建614.2.2AC6 BC4F2分离群体的表型鉴定624.2.3目标QTL区段连锁图谱构建634.2.4AC6的遗传效应分析644.2.5AC6区段标记开发及其精细定位654.2.6候选基因的预测684.3讨论694.3.1QTL标记辅助选择对近等基因系构建的有效性694.3.2近等基因系BC4F2分离群体与F2:3定位结果的比较704.3.3AC6精细定位效果70第五章 玉米叶脉、叶耳颜色主效QTL-AP10近等基

7、因系的构建及进一步定位735.1材料与方法735.1.1试验材料735.1.2试验方法735.2结果与分析745.2.1AC10近等基因系的构建745.2.2AC10 BC4F2分离群体的表型鉴定755.2.3AC10区段标记开发765.2.4目标QTL区段连锁图谱构建765.2.5AC10的遗传效应分析765.3讨论77第六章 玉米籽粒花色苷含量两个显性基因AC6和AC10的互作效应分析796.1材料与方法796.1.1试验材料796.1.2试验方法796.2结果与分析806.2.1BC4F2基因型和表型分析806.2.2AC6和AC10的加性、显性及上位性效应估算826.3讨论83第七章

8、主要结论和主要创新点857.1主要结论857.1.1玉米花色苷含量和Fe、Zn含量相关性状的QTL初步定位857.1.2玉米籽粒中花色苷含量主效QTLAC6的精细定位和候选基因预测857.1.3玉米籽粒中的花色苷含量主效QTL AC10的验证和进一步准确定位867.1.4AC6和AC10的上位性互作研究867.2主要创新点867.2.1玉米籽粒花色苷含量目标基因的创新867.2.2玉米籽粒和穗轴中Fe、Zn含量的QTL定位87参考文献89致谢91发表论文及参加课题一览表93玉米花色苷含量相关性状和铁 (Fe)、锌 (Zn)含量相关性状的QTL初步定位及玉米籽粒花色苷含量两个主效QTL AC6和

9、AC10的精细定位和上位性研究摘 要玉米是世界上分布最广的作物，也是我国种植面积最大的粮食作物。随着生活水平的不断提高和饮食结构的调整，人们对玉米的营养品质要求越来越高。其功能性品质作为重要的营养品质之一，对防治疾病、增进人体健康起着非常重要的作用，是开发天然保健食品的重要原料，同时也广泛应用于医药、化妆品等行业中。黑（紫）玉米中富含的花色苷和微量元素对机体具有多种生理生化功能。因此，黑玉米是研究花色苷和Fe、Zn等矿质元素代谢的良好材料。认识花色苷合成和矿质元素平衡的遗传规律，可以为我国玉米的功能性品质在天然保健食品及在医药、化妆品等领域中的应用提供新的参考和借鉴。西南大学玉米研究所新创了一

10、个新的特异的黑玉米材料SDM (super dark maize)，本研究利用SDM为共同父本，分别与白玉米自交系木6和黄玉米自交系Mo17杂交，构建两套相关F2:3群体（MuS-F2:3和MoS-F2:3），在重庆歇马和云南元江两个环境中种植F2:3家系，分别对玉米中的15个花色苷含量相关性状及玉米籽粒和穗轴中Fe、Zn含量4个性状进行了初步QTL分析。并在此基础上利用高世代回交群体（BC4F2）对玉米籽粒中的花色苷含量主效基因之一AC6进行了精细定位，对另一个主效基因AC10也进行了验证和进一步准确定位，并对这两个主效基因的上位性互作进行了初步分析。以期为玉米中花色苷和Fe、Zn含量的相关

11、研究提供新的参考与借鉴，为高花色苷和Fe、Zn含量玉米资源的鉴定及基因工程奠定理论基础，为高花色苷含量和Fe、Zn含量分子标记辅助选择和育种改良提供依据。主要研究结果如下：1、 亲本及杂交后代的花色苷含量相关性状的表型特征共同父本SDM的籽粒、穗轴、叶耳、叶脉、叶鞘、花药、花药护颖等七个部位的颜色均表现为紫色，Mo17和木6表现为普通玉米常见的非紫色。SDM籽粒和穗轴中的花色苷含量、黑色素含量、总酚含量均极显著高于Mo17和木6，类黄酮含量SDM与Mo17和木6差异均不显著。两个相关F2:3群体的表型表现一致：均表现为连续分布，但不完全符合严格的正态分布，偏度和峰度均较大，说明花色苷含量相关性

12、状均为数量性状，且受主效基因控制。所有花色苷含量相关性状的遗传力均高于75%。两个群体表型相关性分析结果高度一致，色素相关性状彼此之间存在较大的正相关。2、 亲本及杂交后代籽粒和穗轴Fe、Zn含量的表型特征SDM籽粒和穗轴中的Fe和Zn含量与Mo17和木6存在极显著差异，SDM籽粒中的Zn含量极显著高于木6，穗轴中的Zn含量以及籽粒和穗轴中的Fe含量均极显著低于木6；SDM籽粒和穗轴中的Zn含量以及籽粒中的Fe含量均极显著高于Mo17，穗轴中的Fe含量极显著低于Mo17。两个群体F2:3家系的表型表现一致：均表现为连续分布，但是偏度和峰度偏高，不是典型的正态分布。相对于色素相关性状，籽粒和穗轴

13、中Zn和Fe含量的遗传力较低，尤其是籽粒中Fe含量的遗传力低于60%，其他三个性状的遗传力大于70%。矿质元素相关性状彼此之间存在一定的相关性，籽粒中的Fe和籽粒中的Zn以及穗轴中的Fe和穗轴中的Zn显著正相关，籽粒中的Fe和穗轴中的Fe以及籽粒中的Zn和穗轴中的Zn不相关。3、 F2群体连锁图谱的构建MuS群体构建了含160个标记位点的玉米基因组的13个连锁群,总的遗传距离为1,298.067 cM, 标记间的平均距离为8.11 cM。每条染色体包含有1124个SSR标记, 平均为16个；MoS群体,构建了含170个标记位点的玉米基因组的12个连锁群,连锁群全长1,104.92 cM, 标记

14、间平均距离为6.46 cM。每条染色体包括1022个SSR标记, 平均为17个。4、 玉米花色苷含量相关性状的QTL定位两个群体分别检测到65个和73个与花色苷含量相关的QTL，分布在除了第5染色体外的9条玉米染色体上。MuS中，8个染色体区段存在环境顿感QTL（即在该区域中存在的QTL在不同环境中均能被检测到），位于第1、6、7、9、10染色体上；MoS中，10个染色体区段存在环境顿感QTL，位于第1、4、6、7、10染色体上。两个群体共检测到5个花色苷含量“一致性QTL”，位于bin1.04, bin4.06, bin6.04, bin7.04和bin10.04。控制不同性状的QTL有成簇

15、分布的现象，主要表现在第6和第10染色体，bin6.04和bin10.04区域内几乎包含控制所有花色苷含量相关性状的QTL，且大多数QTL对表型的贡献率大于10%，因此认为这两个染色体区段存在控制花色苷含量相关性状的主效基因。5、 玉米籽粒和穗轴中Fe、Zn含量的QTL定位共检测到32个与籽粒和穗轴中Fe、Zn含量相关的QTL，重庆和云南两个环境中分别检测到17个和15个。在MuS和MoS中分别发现3个和4个染色体区域存在环境钝感QTL，位于第2、7、9、10染色体上。两个群体检测到的QTL在标准图谱（IBM2 2008 Neighbors Frame6）上进行整合，结果表明位于第2、7、9三

16、条染色体上的QTL在两个群体中均能检测到，而且位置和效应高度一致。位于这三条染色体上的控制不同性状的QTL有成簇分布的现象，主要表现在第2染色体上检测到的QTL同时与ZnK, ZnC, FeK 和FeC有关，第9染色体上检测到的QTL同时与Znk, FeK和FeC有关，第7染色体上检测到的QTL同时与ZnK和ZnC有关，成簇分布的QTL可能是紧密连锁也可能是一因多效的同一QTL，从遗传学的层面解释了不同性状间表型的相关性。6、 玉米籽粒中的花色苷含量主效基因AC6的精细定位及候选基因预测在MuS-BC4F2和MoS-BC4F2中，分别用6对和7对多态性SSR标记构建了目标QTL区段的遗传连锁图

17、谱，结合籽粒中的花色苷含量，对第6染色体上控制玉米籽粒花色苷含量的主效QTL（命名为AC6）进行了验证，两个群体中分别将其进一步定位到了标记区间umc1857-umc1014和umc1979-umc1014。在该区段内自行设计SSR标记，将AC6精细定位在了SSR标记S8和umc1014之间，这两个标记位于bin6.04区的ctg280上，两者之间物理距离518Kb。该区间的相应位置存在一个Z576C20.2（PL transcription factor）血红素加氧酶基因，参与玉米色素调节。为了确定Z576C20.2是否是候选基因，设计了1对引物对BC4F2世代中目的性状对应的极端株系（目标

18、表型与SDM最一致的株系视为目标性状的“突变体”（SDMm），与木6或Mo17性状一致的视为“野生型”（SDMw）的Z576C20.2基因进行扩增、测序。测序结果发现，该基因的碱基序列未发生任何变化。推测该突变性状可能是由其它基因发生突变或者是由该基因启动子序列发生突变造成。7、 玉米籽粒中的花色苷含量主效基因AC10的验证和进一步准确定位在MuS-BC4F2群体中，加大标记密度，构建了包含7对SSR标记的目标区段遗传连锁图谱，结合基因型和表型，对第10染色体上的另一个与玉米籽粒中花色苷含量相关的主效QTL（命名为AC10）进行了进一步定位。叶脉颜色检测到了两个QTL位点，分别位于标记区间ID

19、P7852-IIDP8526和S44-bnlg1028内，叶耳颜色检测到了一个QTL，位于区间S44-bnlg1028内。籽粒花色苷含量没有检测到QTL，可见第10染色体上的基因不能单独控制玉米籽粒花色苷的含量。目的基因所在染色体区段内，很难找到亲本多态性标记。因此，要想在此基础上对AC10开展进一步的精细定位，找到足够的多态性标记是关键。8、 AC6和AC10的上位性互作研究通过对BC4F2双片段聚合系基因型和表型的分析，以及对两个基因加性、显性和上位性的估算结果，表明AC6和AC10单座位QTL的加性效应和显性效应都较小，AC6单座位的加性效应和显性效应相对较大。AC10单独存在时对增大玉

20、米籽粒中的花色苷含量没有显著作用。当AC6和AC10同时存在时，两个基因会发生互作，使得玉米籽粒中的花色苷含量大大提高。关键词：玉米；花色苷含量；铁（Fe）含量；锌（Zn）含量；QTL初步定位；QTL精细定位；上位性分析QTL analysis of anthocyanin-related traits and Fe and Zn concentration-associated traitsand fine mapping and analyzing epistatic gene effects of two genes AC6 and AC10 that controls anthocya

21、nin content in maize(zea mays L)kernelPh.D. Candidate:HongniQinSupervisor: Prof. YilinCaiAbstractMaize(zea mays L) is an important kind of grain crop which covers the biggest area in China with widest distribution in the world. With living standars improvement and diet adjustments, pople have more a

22、nd more demands on nutrient quality of maize. The functional quality of maize plays a significant rule to prevent diseases and promote the health of human body. Black (purple) maize is a good source of anthocyanins and microelements, which could be regarded as an excellent material for genetic and m

23、etabolic research of anthocyanin, iron and zinc. Exploration of intrinsic genetic law on molecular level for biosynthesis of anthocyanin and homeostasis of microelement could provide references and useful guidance for black maize future applications in medicine, cosmetics and other fields.A newly bl

24、ack-maize inbred line SDM (Super Dark Maize) bred by Maize Research Institute of Southwest Univercity was used as a common male parent to cross with Mu6 (white kernel) and Mo17 (yellow kernel) respectively. Four datasets from two F2:3 populations (MuS-F2:3 and MoS-F2:3) and two environments (Chongqi

25、ng and Yunnan) were used to analyze the QTLs for 15 anthocyanin-related traits and 4 Fe and Zn conventration-sssociated traits.Fine mapping and analyzing epistatic gene effects of two genens AC6 and AC10 that controls anthocyanin content in maize kernel was conducted based on the preliminary QTL map

26、ping. We expect providing basis and useful guidance for further study of biosynthesis of anthocyanin and homeostasis of microelement. The main results were as follows: 1. Phenotype performance of anthocyanin-related traits for F2:3 families and their parentsThe colors of kernel, cob, auricle, leaf v

27、ein, leaf sheath, anther and anther-cape of male parent SDM were all daker (dark purple) than female parents Mu6 and Mo17, and the anthocyanin, melanin, total phenolic contents of SDM kernel and cob were also statistically higher than Mu6 and Mo17. There were no significant difference in flavonoid c

28、ontent between SDM and Mu6/Mo17. All traits of F2:3 families continuously segregated but not normally distributed with higer kurtosis and skewness, which indicated that all traits were quantitative trait loci (QTL) controlled by combination with multiple-gene and maior gene. The heritabilities of al

29、l traits were more than 0.75. In both F2:3 populations, most of traits had significant positive correlation to each other.2. Phenotype performance of Fe, Zn concentraition-related traits for F2:3 families and their parentsThere were highly significant differences between two parents in ZnK, FeK, ZnC

30、 and FeC in two populations. In MuS population, ZnK of SDM was highly significantly higher than Mu6, whereas ZnC, FeK, FeC of SDM were highly significantly lower than Mu6. In MoS population, ZnK, ZnC and FeK of SDM were highly significantly higher than Mo17, whereas FeC of SDM was highly significant

31、ly lower than Mo17. The frequency distributions of the four traits showed continuous phenotypic variation, but all four traits were not normally distributed. The heritability of FeK (0.6) was lower than the heritability of other three traits (0.7). There existed significant or highly significant pos

32、itive correlations between ZnK and FeK, ZnC and FeC, whereas ZnK and FeK did not significantly correlate to ZnC and FeC.3. Linkage map construction for the two F2 populationsFor MuS population, 160 polymorphism markers were used to develop the genetic map. These SSR loci were mapped on 13 linkage gr

33、oups covering 1,298.067 cM of the whole genome with an average interval of 8.11 cM between adjacent markers. The average number of markers per chromosome was 16 ranged from 11 in chromosome 7 to 24 in chromosome. For MoS population, 171 polymorphism markers were used to construct the genetic map. Th

34、ese SSR loci were placed on 12 linkage groups representing the 10 maize chromosomes. The genetic map spanned 1,104.92 cM in length with an average interval of 6.46 cM between adjacent markers. The number of markers ranged from 10 in chromosome 7 to 22 in chromosome 1 with the average number of 17 ma

35、rkers per chromosome. 4. QTL mapping for anthocyanin-related traitsTotally 65 and 73 QTLs were detected for MuS-F2:3 and MoS-F2:3, redpectively, which were distributed on chromosome 1, 2, 3, 4, 6, 7, 8, 9 and 10. It was found that eight and ten QTLs detected in different environments for the two pop

36、ulations respectively, were located in the same genetic regions, and these stable QTLs were distributed on chromosome 1, 4, 6, 7, 9 and 10. Through the comparison of the two populationsresults, five congruent QTLs were identified for anthocyanin-related traits in bin1.04, bin4.06, bin6.04, bin7.04 a

37、nd bin10.04 respectively. Furthermore, the distribution of QTLs for these traits showed a high concentration of QTLs in few chromosome regions. A number of QTLs which were located in bin6.04 and bin10.04 were clustered. For most of these QTLs, the contribution rate of phenotypic variation was more t

38、han 10%. The two chromosome regions were considered to have major genes mainly controlling anthocyanin-ralated traits.5. QTL mapping for Fe, Zn conventration-related traitsA total of16 and 15 QTLs were identified in CQ and YN respectively, some of which were identical in different environments. The

39、common regions for same trait at different environments were 3 and 5 in MuS-F2:3 and MoS-F2:3 respectively. Compared with the IBM2 2008 Neighbors Frame6, the distribution and effect of some QTLs in two populations were highly consistent and many QTLs on chromosome 2, 7 and 9 were detected in both po

40、pulations. Moreover, several mineral QTLs co-localized with each other for both populations such as the QTLs for ZnK, ZnC, FeK and FeC on chromosome 2, QTLs for Znk, FeK and FeC on chromosome 9 and QTLs for ZnK and ZnC on chromosome 7, which probably were closely linked to each other, or were the sa

41、me pleiotropic QTL. The co-localizations may explain the phenotypic correlations among traits.6. Fine mapping of main QTL AC6 that control anthocyanin content of maize kernel and prediction for candidate genesFor BC4F2-MuS and BC4F2-MoS, a total of 6 and 7 polymorphic SSR markers were used to constr

42、uct linkage map of target region on chromosome 6 (bin 6.04), respectively. A major gene AC6related to anthocyanin content of maize kernel was further located in the interval umc1857-umc1014 and umc1979-umc1014 in BC4F2-MuS and BC4F2-MoS respectively. To fine map the gene AC6, the self-designed prime

43、rs for the target gene were used and AC6 was mapped between S8 and umc1014with genetic distance 518Kb. In this region, a heme add oxygen enzyme gene Z576C20.2 (pltranscription factor), which is a transcription factor involved in the regulation of maize pigment. The Z576C20.2 of two extreme individua

44、ls of BC4F2 (“mutant plant” (SDMm) and “wild plant” (SDMw) were amplified, sequenced and compared. The PCR products electrophoretic patterns and base sequence of SDMm and SDMw were completely concordant. The results showed Z576C20.2 was not the candidate gene of AC6. Perhaps, the high anthocyanin co

45、ntent trait may be controlled by other gene or caused by promoter mutation of Z576C20.2. 7. Further mapping of the other QTL AC10 that control anthocyanin content of maize For BC4F2-MuS, a total of 7 polymorphic SSR markers were used to construct linkage map of target region on chromosome 10 (bin 10

46、.04). Two QTLs related to COV (colur of leaf vein) were further located in the interval IDP7852-IIDP8526 and S44-bnlg1028. One QTL related to COA (colur of leaf auricle) was fuether mapped in the intervalS44-bnlg1028. No QTL related to ACK (anthocyanin content of kernel) was detected. In order to fi

47、ne mapping the major gene AC10,enoughpolymorphic marker is crucial.8. Epistasis analysis of AC6 and AC10Double segment substitution line (DSSL) of AC6 and AC10 was used to analyze additive, dominant and epistatic effects of the two major QTLs. The additive effect and dominant effect of AC6 was relatively greater than AC10,but the epistatic effects of AC6 and AC10played a major role in anthocyanin content of maize kernel.Key words: maize (zea mays L);anthocyanin content; iron (Fe) concentration; zinc (Zn) concentration; QTL preliminary map; QTL fine map;